EC Number | Cloned (Comment) | Organism |
---|---|---|
1.1.1.28 | gene ldhD, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Lactobacillus delbrueckii subsp. bulgaricus |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
1.1.1.28 | F299Y | site-directed mutagenesis | Lactobacillus delbrueckii subsp. bulgaricus |
1.1.1.28 | additional information | highly stereoselective biosynthesis of (R)-alpha-hydroxy carboxylic acids through rationally re-designed mutation of D-lactate dehydrogenase, asymmetric reduction of a homologous series of alpha-keto carboxylic acids such as phenylpyruvic acid, 2-oxobutyric acid, 2-oxovaleric acid, beta-hydroxypyruvate, overview. Compared with wild-type D-nLDH, the Y52L mutant D-nLDH shows elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-alpha-hydroxy carboxylic acids can be produced at high yields and highly optical purity. Production of chiral (R)-phenyllactic acid. 50 mM PPA is completely reduced to (R)-phenyllactate in 90 min with a high yield of 99.0% and a highly optical purity (99.9% e.e.) by the engineered coupled production system. Activties of the F299Y mutant are similar to the wild-type enzyme | Lactobacillus delbrueckii subsp. bulgaricus |
1.1.1.28 | Y52L | site-directed mutagenesis | Lactobacillus delbrueckii subsp. bulgaricus |
1.1.1.28 | Y52L/F299Y | site-directed mutagenesis | Lactobacillus delbrueckii subsp. bulgaricus |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.1.1.28 | 0.27 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L | Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | 0.32 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | 1.4 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L/F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | 11.4 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged wild-type enzyme | Lactobacillus delbrueckii subsp. bulgaricus |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.1.1.28 | (R)-lactate + NAD+ | Lactobacillus delbrueckii subsp. bulgaricus | - |
pyruvate + NADH + H+ | - |
r | |
1.1.1.28 | (R)-lactate + NAD+ | Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | - |
pyruvate + NADH + H+ | - |
r | |
1.1.1.28 | pyruvate + NADH + H+ | Lactobacillus delbrueckii subsp. bulgaricus | - |
(R)-lactate + NAD+ | - |
r | |
1.1.1.28 | pyruvate + NADH + H+ | Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | - |
(R)-lactate + NAD+ | - |
r |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
1.1.1.28 | Lactobacillus delbrueckii subsp. bulgaricus | - |
- |
- |
1.1.1.28 | Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | - |
- |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
1.1.1.28 | recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) to homogeneity by nickel affinity chromatgraphy | Lactobacillus delbrueckii subsp. bulgaricus |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.1.1.28 | (R)-lactate + NAD+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | pyruvate + NADH + H+ | - |
r | |
1.1.1.28 | (R)-lactate + NAD+ | - |
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | pyruvate + NADH + H+ | - |
r | |
1.1.1.28 | 2-oxobutyrate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | 2-hydroxybutyrate + NAD+ | - |
? | |
1.1.1.28 | 2-oxobutyrate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | 2-hydroxybutyrate + NAD+ | - |
? | |
1.1.1.28 | 2-oxovalerate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | 2-hydroxyvalerate + NAD+ | - |
? | |
1.1.1.28 | 2-oxovalerate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | 2-hydroxyvalerate + NAD+ | - |
? | |
1.1.1.28 | additional information | residues Tyr52 and Phe299 are mainly responsible for hindering larger substrates because of their short distance from the side chain of 2-oxocarboxylic acids and their steric orientation in the wild-type enzyme. Substrate specificity and enantioselectivity of D-nLDH and DnLDH mutants, overview | Lactobacillus delbrueckii subsp. bulgaricus | ? | - |
? | |
1.1.1.28 | additional information | residues Tyr52 and Phe299 are mainly responsible for hindering larger substrates because of their short distance from the side chain of 2-oxocarboxylic acids and their steric orientation in the wild-type enzyme. Substrate specificity and enantioselectivity of D-nLDH and DnLDH mutants, overview | Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | ? | - |
? | |
1.1.1.28 | phenylpyruvate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | D-phenyllactate + NAD+ | - |
? | |
1.1.1.28 | pyruvate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | (R)-lactate + NAD+ | - |
r | |
1.1.1.28 | pyruvate + NADH + H+ | - |
Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 | (R)-lactate + NAD+ | - |
r |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
1.1.1.28 | D-nLDH | - |
Lactobacillus delbrueckii subsp. bulgaricus |
1.1.1.28 | NAD-dependent D-lactate dehydrogenase | - |
Lactobacillus delbrueckii subsp. bulgaricus |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
1.1.1.28 | 37 | - |
assay at, 2-oxoacid reduction activity | Lactobacillus delbrueckii subsp. bulgaricus |
EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.1.1.28 | 1.8 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | 11.3 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged wild-type enzyme | Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | 1447 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L/F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | 2013 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L | Lactobacillus delbrueckii subsp. bulgaricus |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
1.1.1.28 | 7.5 | - |
assay at, 2-oxoacid reduction activity | Lactobacillus delbrueckii subsp. bulgaricus |
EC Number | Cofactor | Comment | Organism | Structure |
---|---|---|---|---|
1.1.1.28 | NAD+ | - |
Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | NADH | - |
Lactobacillus delbrueckii subsp. bulgaricus |
EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.1.1.28 | 1 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged wild-type enzyme | Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | 5.7 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | 1000 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L/F299Y | Lactobacillus delbrueckii subsp. bulgaricus | |
1.1.1.28 | 7500 | - |
phenylpyruvate | pH 7.4, 37°C, recombinant His-taged mutant Y52L | Lactobacillus delbrueckii subsp. bulgaricus |